JAK3-SH2 domain targeting drugs for breast cancer treatment

Abstract

Provided herein are allosteric compounds, inhibitors or inhibitor compounds selective for the kinase domain or the non-kinase domains Jak3-SH2 or FERM-SH2 in the Jak3 enzyme. Also provided are methods for treating a breast cancer, such as a triple negative breast cancer and it brain metastases, for preventing or reducing the likelihood of the triple negative brain cancer metastasizing, and for inhibiting the proliferation of cancer cells from, for example, a breast cancer, a uterine cancer and an ovarian cancer or their metastases.

Description

[0001] JAK3-SH2 DOMAIN TARGETING DRUGS FOR BREAST CANCER TREATMENT

[0002] Cross-Reference to Related Applications

[0003] This international patent application claims benefit of priority under 35 U.S.C. §119(e) of provisional patent application U.S. Serial No. 63 / 652,511 , filed May 28, 2024, the entirety of which is hereby incorporated by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Field of the invention

[0006] The present invention relates generally to the fields of drug discovery and breast cancer treatment. More specifically, the present invention relates to the treatment of the triple negative breast cancer subtype and its brain metastases with drugs targeting the Janus Kinase 3-Src homolog domain (Jak3-SH2).

[0007] Description of the Related Art

[0008] Breast cancer is the most common malignancy in women where triple negative breast cancer (TNBC), accounts for more breast cancer-related deaths. The patients with triple negative breast cancer subtype of breast cancer represent only 15-25% of total breast cancer with the shortest time to metastasis development and show the shortest survival time of only 4.9 months. Overall, nearly 50% of patients with triple negative breast cancer develop brain metastasis during their course of the disease progression. Interestingly, this subtype of breast cancer is reported to respond well to the chemotherapeutic agents combined with whole brain radiation therapy (WBRT) as compared to the other types of breast tumors.

[0009] In triple negative breast cancer, the cancer cells lack the expression of three important receptors (ER: Estrogen receptor, PR; Progesterone Receptor, and HER2: human epidermal growth factor receptor 2). In the absence of these receptors-mediated tyrosine kinase signaling, the triple negative breast cancer cells adopt alternate kinase mediated signaling including Janus Kinases (Jak) to survive and maintain cell proliferation and facilitate metastasis. Jak3 regulates epithelial cell proliferation and cytoskeletal remodeling, and both these processes are essential for triple negative breast cancer cell proliferation and metastasis.

[0010] T riple negative breast cancer cells have a significantly higher brain metastatic rate and currently used chemotherapeutic agents used in treating brain metastasized triple negative breast cancer have poor blood brain permeability. This in turn necessitates the use of whole brain radiation therapy which significantly increases radiation associated neurotoxicity and neurological complications in these patients. Thus, there is an unmet need in blocking triple negative breast cancer cell proliferation and metastasis in cancer patients through Jak3- directed anticancer agents which in-turn could prevent / suppress cancer proliferation, brain metastasis, and neurotoxicity.

[0011] There is a need in the art for selective therapeutics to treat a breast cancer with decreased toxicity and to prevent advancement of a metastatic stage. Specifically, there is a need for methods to treat a triple negative breast cancer and its metastatic brain cancer with allosteric compounds selective for a Janus kinase 3-Src homolog 2 domain in the Jak3 enzyme.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is directed to an allosteric compound that selectively targets a non-kinase domain in Janus Kinase 3 enzyme (Jak3). Particularly, the non-kinase domain is a Jak3-Src homology 2 (Jak3-SH2) domain.

[0014] The present invention is further directed to a pharmaceutical composition comprising the allosteric compound described herein and a pharmaceutically acceptable diluent or excipient.

[0015] The present invention is directed further to a method for treating a breast cancer in a patient in need thereof. In this method, an amount of the pharmaceutical composition described herein pharmacologically effective to inhibit at least one activity of a Jak3 enzyme is administered at least once to the patient.

[0016] The present invention is directed further still to a method for preventing or reducing the likelihood of a triple negative brain cancer metastasizing in a subject. In the method a pharmaceutically effective amount of N-methylsulfonylmethanesulfonamide or a pharmaceutical composition thereof is administered at least once to the subject.

[0017] The present invention is directed further still to a method for inhibiting proliferation of cancer cells overexpressing a Janus kinase 3 enzyme. In this method, the cancer cells are contacted with at least one inhibitor selective for a Jak3-SH2 domain in the Janus kinase 3 enzyme.

[0018] The present invention is directed further still to an inhibitor compound selective for a specific domain in a Janus Kinase 3 (Jak3) enzyme. Particularly, the specific domain is the kinase domain, the Jak3-SH2 domain or the FERM-SH2 domain.

[0019] The present invention is directed further still to a pharmaceutical composition comprising the inhibitor compound described herein and a pharmaceutically acceptable diluent or excipient. The present invention is directed further still to a method for treating a breast cancer in a patient in need thereof. In the method administering at least once to the patient an amount of the inhibitor described herein or a pharmaceutical composition thereof is administered at least once to the patient, either of which is pharmacologically effective to inhibit at least one activity of the Jak3 enzyme.

[0020] Other and further aspects, features, benefits, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The appended drawings have been included herein so that the above-recited features, advantages, and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.

[0023] FIGS. 1A-1 B show a schematic of the full-length JAK3 and its kinase and non-kinase domain structure (FIG. 1A) and illustrates the ATP binding domain of Jak3 (circled) used as the target to develop highly selective Jak3 inhibitors (FIG. 1 B).

[0024] FIGS. 2A-2C show Al-based smart druggable site screening for Jak3-targeted drugs where virtual compounds binding to human Jak 3 co-crystalized with a smart drug. FIG. 2A illustrates a co-crystalized image and FIG. 2B illustrates the modeling site defined by the following residues in the structure amino-acid residues in Jak3 binding with inhibitor. FIG. 2C illustrates an Al-based virtual screening fort he druggable sites in Jak3 through binding of virtual compounds.

[0025] FIG. 3 shows that expression of pJak3 (red) decreases with the C1 and C4 compound treatment.

[0026] FIG. 4 shows that expression of pStat-3 (red) decreases with the C1 and C4 compound treatment in the TNBC-BM cell line.

[0027] FIGS. 5A-5B show the overexpression of Jak3 in brain biopsies of human TNBC patients (FIG. 5A) and in the TNBC-BM cell specific cell line (FIG. 5B).

[0028] FIGS. 6A-6I show an increase in Jak3 expression in uterine sarcoma (FIGS. 6A-6C), in uterine endometrial cancer (FIGS. 6D-6F) and in ovarian cancer (FIGS. 6G-6I).

[0029] FIGS. 7A-7B show the effects of compounds C1 and C4 on Jak 3 phosphorylation (FIG. 7A) and cell proliferation (FIG. 7B) on TNBC cell lines.

[0030] FIGS. 8A-8D shows that Jak3-targeted small molecule inhibitors (C1 and C4) have anti-TNBC-brain metastasis. FIG. 8A shows the time-course of the growth of tumor volume at the primary site of breast tissue. FIGS. 8B-8C show representative confocal images for Jak3 (upper) and Vimentin (brain metastatic marker) (FIG. 8B) in brain metastases of mice treated with C1 (FIG. 8C). The scale bar represents 100 pm. Bar graph comparing mice treated with PBS or C1 before and after intraperitonially injection of MDA-231-BrM-831 cells. n=5 each. *p<0.05. FIG. 8D are confocal images showing the decrease in expression of Jak3 after C1 treatment.

[0031] FIGS. 9A-9D show a Jak3-specific dose response in a cell culture model of triple negative breast cancer-brain metastases (TNBC-BM).

[0032] FIGS. 10A-1 OB show that C1 fails to inhibit other Jak kinases in vivo in a Jak3-deficient mouse.

[0033] FIG. 11 shows blood brain barrier permeability in vitro using a trans-well cell culture of endothelial and astrocyte cells.

[0034] FIGS. 12A-12B show that C1 stimulates a tumorigenic immune response in vivo.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] As used herein, the articles "a" and "an" when used in conjunction with the term “comprising” in the claims and / or the specification, may refer to “one”, but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”. Some embodiments of the invention may consist of or consist essentially of one or more elements, components, method steps, and / or methods of the invention. It is contemplated that any composition, component or method described herein can be implemented with respect to any other composition, component or method described herein.

[0037] As used herein, the term “or” in the claims refers to “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or”.

[0038] As used herein "another" or “other” may mean at least a second or more of the same or different claim element or components thereof.

[0039] As used herein, the terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included.

[0040] As used herein, the terms "consist of" and "consisting of" are used in the exclusive, closed sense, meaning that additional elements may not be included.

[0041] As used herein, the term “includes” or “including” refers to “including, but not limited to”. The terms “includes, “including” and “including, but not limited to” are used interchangeably.

[0042] As used herein, the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., ± 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.

[0043] As used herein, the term “allosteric compound” refers to a therapeutic compound that targets only the Jak3-SH2 domain in a Janus kinase 3 enzyme to selectively inhibit or interfere with at least one activity thereof, such as, but not limited to, overexpression and / or a signaling pathway in a cancer or a metastasis thereof associated with Jak3 overexpression.

[0044] As used herein, the term “therapeutically effective amount” refers to a dosage sufficient to achieve a measurable improvement in the breast cancer and / or its metastatic cancers or other cancers and metastases thereof associated with Jak3 overexpression or progression, though not necessarily a cure.

[0045] As used herein, the term “pharmaceutically acceptable” refers to a composition that is safe and suitable for use in contact with cells and tissues in accordance with established medical practices.

[0046] As used herein, the term “treatment” refers to interventions aimed at alleviating, preventing, stabilizing, or resulting in remission of a cancer, for example, but not limited to, breast cancer and / or its metastatic cancers or other cancers and metastases thereof associated with Jak3 overexpression.

[0047] As used herein, the terms “allosteric compound”, “inhibitor” and “inhibitor compound” are used interchangeably.

[0048] As used herein, the terms “inhibit”, “inhibitor" or “inhibitor compound" denotes a reduction in activity or a compound effective to enable a reduction in activity, for example, in JAK3 activity, such as, but not limited to, autophosphorylation activity or preventing or reducing overexpression of the enzyme or signaling pathways associated therewith, which may range from partial to complete suppression. In certain embodiments, inhibition refers to a decrease of at least 10%, 20%, 50%, or even 100% compared to control levels.

[0049] As used herein, the compound terms “Al-C#4”, “Al-C#14”, “Al-C#15”, “Al-C#17”, “Al- C#18”, “Al-C#19”, “Al-C#22”, and “Al-C#23” are interchangeable.

[0050] As used herein, the compound terms “SH2-C1”, “SH2-C2”, “SH2-C3”, “SH2-C4”, and “SH2-C5” and “C1”, “C2”, “C3”, “C4”, and “C5” are interchangeable.

[0051] As used herein, the term “contacting” refers to any suitable method of bringing a compound or a pharmaceutical composition into contact with a cell in vivo, in vitro or ex vivo. For in vivo applications, any known method of administration is suitable as described herein.

[0052] As used herein, the term “subject", refers to a human that is the recipient of the allosteric compounds or inhibitors described herein or is a control. As used herein, the term “patient", refers to a subject who is undergoing clinical treatment for a cancer overexpressing Jak3 enzyme and / or it metastases, such as, but not limited to, triple negative breast cancer and / or its metastases.

[0053] In one embodiment of the invention, there is provided an allosteric compound that selectively targets a non-kinase domain in Janus Kinase 3 enzyme (Jak3).

[0054] In this embodiment, the non-kinase domain may be a Jak3-Src homology 2 (Jak3- SH2) domain. Also in this embodiment, the allosteric compound may be N- methylsulfonylmethanesulfonamide, N-(1 -cyano-1 -methyl-ethyl)-2-methoxy-ethanesulfon amide, N-(2-cyanophenyl)methyl-N-ethyl-ethanesulfonamide, N-(2-cyanoethyl)-N-methyl- propane-1 -sulfonamide, or 5-methyl-2-prosylsulfonyl-benzamide or a combination thereof. Particularly, the allosteric compound is N-methylsulfonylmethanesulfonamide.

[0055] In another embodiment of the invention, there is provided a pharmaceutical composition comprising the allosteric compound as described supra and a pharmaceutically acceptable diluent or excipient.

[0056] In yet another embodiment of the invention, there is provided a method for treating a breast cancer in a patient in need thereof, comprising administering at least once to the patient an amount of the pharmaceutical composition as described supra pharmacologically effective to inhibit at least one activity of a Jak3 enzyme.

[0057] In this embodiment, the administering step may comprise selectively targeting a Jak3- SH2 domain within the Jak3 enzyme. Also in this embodiment, the pharmaceutical composition may comprise N-methylsulfonylmethanesulfonamide and the diluent or the excipient. In addition, the activity of the Jak3 enzyme may be a signaling pathway within cells associated with the breast cancer. Furthermore, the breast cancer may a triple negative breast cancer (TNBC) or a metastatic cancer thereof. Particularly, the metastatic cancer is a metastatic brain cancer.

[0058] In yet another embodiment of the invention there is provided a method for preventing or reducing the likelihood of a triple negative brain cancer metastasizing in a subject, comprising administering at least once to the subject a pharmaceutically effective amount of N-methylsulfonylmethanesulfonamide or a pharmaceutical composition thereof. In this embodiment, the triple negative breast cancer may metastasize to a metastatic brain cancer.

[0059] In yet another embodiment of the invention, there is provided a method for inhibiting proliferation of cancer cells overexpressing a Janus kinase 3 enzyme, comprising contacting the cancer cells with at least one inhibitor selective for a Jak3-SH2 domain in the Janus kinase 3 enzyme.

[0060] In this embodiment, the contacting step may comprise inhibiting at least one signaling pathway in the cancer cells. Also in this embodiment, the inhibitor may be at least one of N- methylsulfonylmethanesulfonamide, N-(1 -cyano-1 -methyl-ethyl)-2-methoxy-ethanesulfon amide, N-(2-cyanophenyl)methyl-N-ethyl-ethanesulfonamide, N-(2-cyanoethyl)-N-methyl- propane-1 -sulfonamide, or 5-methyl-2-prosylsulfonyl-benzamide or a combination thereof. Particularly the inhibitor is N-methylsulfonylmethanesulfonamide. In addition, in this embodiment, the cancer cells may comprise a breast cancer, a uterine sarcoma, a uterine endometrial cancer, or an ovarian cancer. An example of a breast cancer is a triple negative breast cancer. In one aspect, the triple negative breast cancer cells are metastatic brain cancer cells.

[0061] In yet another embodiment of the invention, there is provided an inhibitor compound selective for a specific domain in a Janus Kinase 3 enzyme (Jak3).

[0062] In one aspect of this embodiment the specific domain may be a kinase domain, where the inhibitor comprises 3-(1-(5-(4-fluorophenyl)pyrazolo 1 ,5-a)pyrimidin-7-yl)piperdin-3-yl) propanamide, 5-(2-(5-fluoro-1 H-1 ,3-benzodiazol-2-yl)pyrrolidine-1 -carbonyl)-n-phenyl pyrimidine-2 -amine, N-cyclopropyl-N-((1 H-indol-5-yl)methyl-6-(4H-1 ,2,4-triazol-4-yl)pyridine- 3-carboxamide, N-(5-)carbam(1 ,5-dimethyl-1 H-pyrazol-4yl)carbamoyl)-ethyl-1 H-pyrazol-4- y I )- 1 -methanesulfonylpiperdine-3-carboxamide, N-(1 -2,6-dichlorophenyl)methyl)-1 H-pyrazol- 3-yl)-5-(l-methyl-1 H-pyrazol-4-yl)-1 ,3,4-thiadiazol-2-amine, 4-(azocane-1 -carbonyl -7-fluoro- 2-(2-methylpyrimidin-5-5-yl)quinoxaline-4-amine, 1-(3-chloro-4-fluorphenyl)-4-(4-(2,5-di methylphenyl)-1 ,3-th iazol-2-yl)-1 H-1 ,2,3-triazol-5-amine, or N-(3-ethoxypropyl)-4(2-(4- methoxyphenoxyl)pyrimidin-5-yl) benzamide, or a combination thereof.

[0063] In another aspect of this embodiment the specific domain may be a Jak3-SH2 domain, said inhibitor comprising N-(1 -cyano-1 -methyl-ethyl)-2-methoxy-ethanesulfonamide, N-(2- cyanophenyl)methyl-N-ethyl-ethanesulfonamide, N-(2-cyanoethyl)-N-methyl-propane-1- sulfonamide, or 5-methyl-2-prosylsulfonyl-benzamide or a combination thereof.

[0064] In yet another aspect of this embodiment the specific domain may be a a FERM-SH2 domain, said inhibitor comprising (25,35)-3-amino-2-hydroxy-pentanedioic acid, 5[(2R)-3- hydroxy-2-(methylamino)-3-oxo-propyl]-1 -methyl-pyrazole-4-carboxylic acid, (3R)-3-amino-3- (1 H-imidazo [4,5-b]pyridin-2-yl)propanoic acid, (2R,3R)-3-amino-2-hydroxy-pentanedioic acid, or 5[(2R)-2-amino-3-hydroxy-3-oxo-propyl]-1-pyrazole-4-carboxylic acid or a combination thereof.

[0065] In yet another embodiment of the present invention there is provided a pharmaceutical composition comprising the inhibitor compound described supra and a pharmaceutically acceptable diluent or excipient.

[0066] In yet another embodiment of the present invention there is provided a method for treating a breast cancer in a patient in need thereof, comprising administering at least once to the patient an amount of the inhibitor of claim 21 or a pharmaceutical composition thereof either of which is pharmacologically effective to inhibit at least one activity of the Jak3 enzyme. In this embodiment the breast cancer may be a triple negative metastatic breast cancer or a brain metastasis thereof or a combination thereof.

[0067] Provided herein are allosteric compounds or drugs, inhibitors or inhibitor compounds designed and developed to selectively bind to a specific domain of the Janus kinase 3 (Jak3) enzyme. These domains are the kinase domain and the non-kinase domains Janus kinase 3- Src homology 2 (Jak3-SH2) and FERM-SH2 and the respective compounds are shown in Tables 1 , 3 and 4. The allosteric drugs, inhibitors and inhibitor compounds are therapeutically effective to inhibit its overexpression and / or an activity of the Jak3 enzyme, for example, autophosphorylation or a signaling pathway in cancer cells, thereby decreasing the side effects caused by nonspecific binding of the drug.

[0068] The allosteric compounds or inhibitors described herein may be pharmaceutical compositions comprising one or more of the aforementioned inhibitors formulated with pharmaceutically acceptable diluents, carriers, excipients, salts, and / or adjuvants as are known in the art. Formulation and route of administration are well-known in the art and may depend on the stage of the lung cancer and therapeutic requirements.

[0069] Also provided are methods for treating a cancer and / or a metastatic cancer associated with overexpression of the Jak3 enzyme in a subject or patient in need of such treatment. Examples of these cancers and metastases thereof are a breast cancer, for example, a triple negative breast cancer and its metastatic brain cancer, a uterine cancer, for example, a uterine sarcoma or uterine endometrial cancer and an ovarian cancer. Further provided are methods for inhibiting the proliferation of cancer cells comprising or associated with these compounds, either in vivo or in vitro, via administration of the allosteric compounds, inhibitors or inhibitor compounds or pharmaceutical compositions described herein or via contacting the cancer cells with these compounds. One of ordinary skill in the art is well-able to determine doses and treatment regimens for a specific subject or cancer patient, particularly, a triple negative breast cancer patient with or with a metastatic brain cancer, depending at least on the age, sex, overall health thereof, progression of the cancer, for example, the presence of metastases, or remission thereof.

[0070] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. EXAMPLE 1

[0071] Drug Design and Development

[0072] Artificial intelligence (Al)-based screening of druggable sites in Jak3 and virtual inhibitor compounds

[0073] Development of highly selective Jak3 directed drug could translate into potential therapeutics for triple negative breast cancer-brain metastases (BM) and that alleviate undesirable side effects and would be safe for the treatment of those cancers that overexpress Jak3 requiring long-term therapy. Al (Artificial Intelligence / Deep neural network) methods were used in designing the highly selective Jak3 directed drug in the accurate prediction of drug protein (Jak3) interactions, ensuring better therapeutic efficacy compounds are screened from large data bases containing millions of compounds.

[0074] The X-ray crystal structures for the kinase domain of JAK3 (FIG. 1 A) were used with the ATP binding site (FIG. 1 B) as the only druggable site, and potential inhibitors were first screened by the virtual screen. Sequence alignment of the ATP binding site for the four JAK family members illustrates that JAK3 has only two residues that could be utilized to design selective inhibitors for Jak3. In other Jak family members like JAK1 , JAK2, and Tyk2, these residues are serine and glycine, while in Jak3 they are cysteine and alanine. Molecular modeling was conducted on the X-ray crystal structure of the human JAK3 co-crystalized with an inhibitor is shown (PDB: 5LWN; FIG. 2A). The modeling site defined by the following residues in the structure (after removal of the co-crystallized inhibitor): LEU828, ALA853, VAL884, GLU903, TYR904, LEU905, CYS909, ARG911 , ASP912, ARG953, ASN954, LEU956 and, ASP967 (FIGS. 2B-2C). A molecular library of several million compounds using Atom Net technology was used for structure-based drug design and discovery. Top scoring compounds were clustered and subsequently filtered for favorable properties to arrive at a final subset of 79 compounds.

[0075] In vitro kinase assay

[0076] Furthermore, these compounds were screen based on their binding kinetics in vitro using an in vitro kinase assay. Eight compounds that has higher binding affinity with Jak3 protein was selected to be tested in the TNBC-BM and metastatic brain cancer cell line (see Table 1 ).

[0077] Determination of IC50 value of the 8 in vitro selected compounds

[0078] Eight potential drug compounds were Al-selected that had a higher binding inhibition affinity with Jak3 protein. The specific structures of these top eight compounds are presented in (Table 1 ). The inhibition constant as determined show that the newly discovered Al-based Jak3 specific top eight novel Jak3 targeted potential drug compounds have an IC50 that ranged from 3.36 nM to 18.3 nM (Table 2).

[0079] TABLE 1

[0080] Specific structures of Jak3-specific eight compounds

[0081] TABLE 2

[0082] Efficacy (IC50) of Al-vetted Jak3-specific top eight drug compounds

[0083] Jak3-SH2 domain targeted new class of allosteric drugs to treat triple negative breast cancerbrain metastasis

[0084] Additional compounds were identified that are targeted to other non-kinase and kinase domain of Jak3 (Table 3) through a molecular docking study.

[0085] TABLE 3

[0086] Additional compounds targeted to non-kinase and kinase domains of Jak3 Molecular Docking simulations were performed with the software of AutoDock. The crystal structure of the published truncated-Jak3 (PDB code: 3PJC) was retrieved from the RCSB Protein Data Bank. The solvent molecules within the protein structure were removed in the docking calculations, and the best ligand pose was chosen according to the docking score. Detailed analysis of molecular binding study revealed that compounds C1 through C5 that target the non-kinase SH2 domain of Jak3 bind to Jak3 protein with higher affinity (Table 4).

[0087] TABLE 4

[0088] Specific structures of Jak3-SH2 domain specific five drug compounds

[0089] These were further validated using screening technology of Jak3 in vitro kinase assay which showed that C1-C5 compounds bind to Jak3 with higher affinity and inhibit its kinase activity (Table 5). Comparative analysis of C1-C5 compounds showed that C4 and C5 have higher antiproliferative activity towards metastatic TNBC as compared to C-2, C-3 and C-5 and the mechanism of cell death was found to be TNBC apoptosis. Jak3 phosphorylation and tube formation (indicative of metastasis) ability was found to be significantly decreased in C1 and C4 compound treated triple negative breast cancer cells, MBR-BM-231 (a brain metastatic triple negative breast cancer).

[0090] TABLE 5 Efficacy (IC50) of SH2-domain targeted allosteric Jak3-inhibitor drug compounds

[0091] Pharmacodynamic studies indicated that the expression of phosphorylated Jak3 (pJak3) shown in red fluorescence in FIG. 3 and the phosphorylation of Jak3-substate STAT3 (pStat-3) shown in red in FIG. 4 was significantly decreased upon treatment with the Jak3- targeted inhibitor drug compound C1 and C4.

[0092] EXAMPLE 2

[0093] Overexpression of Jak3 in breast cancer, ovarian cancer and uterine cancer

[0094] FIGS. 5A-5B show, respectively, the overexpression of Jak3 in brain biopsies of human triple negative breast cancer patients, and in the triple negative breast cancer-brain metastases cell specific cell line. Testing was successful in the in vitro efficacy of the drugs in using recombinant Jak3 protein and determining their binding kinetics. Triple negative breast cancer cell line (MDA-MB-231 ), triple negative breast cancer-brain metastatic cell line (MDA-MB-231 -BR) and the MDA-MB-231 -LM control cell line. FIGS. 6A-6I illustrate the overexpression of Jak3 in other cancers specific to women via a comparison of mean Jak3 fluorescence intensity in healthy and cancerous tissues. The results demonstrate a significant increase in Jak3 expression of about 133% between a healthy uterus and a uterine sarcoma (FIGS. 6A-6C), of about 67% between normal uterine tissue and uterine endometrial cancer (FIGS. 6D-6F) and of about 60% between healthy ovaries and cancerous ovarian tissue (FIGS. 6G-6I).

[0095] EXAMPLE 3

[0096] Testing of compounds in vitro and in vivo

[0097] In vitro testing of the compounds in TNBC-BM cell specific cell line

[0098] Triple negative breast cancer cell lines are treated with a control and compounds C#4, C#14, C#15, C#17, C#18, C#19, C#22, and C#23 and tested for Jak3 phosphorylation. Jak3 phosphorylation in triple negative breast cancer cell lines treated with compounds C#1 and C#4 is decreased significantly (FIG. 7A). The compounds were tested as inhibitors of triple negative breast cancer metastasis and triple negative breast cancer brain metastasis cell line through an MTT cell proliferation assay (FIG. 7B).

[0099] In vivo compound testing in mouse xenograph model

[0100] Compounds C#14, C#15, C#17, C#18 are examined in the in vivo mouse xenograft study. The selected compounds are dissolved in buffer and are injected intraperitoneally into the mice at increasing concentrations and are observed for loss of body weight, signs of fatigue, and loss of appetite (food intake). H&E staining of brain sections, microscopic observation for tumors in the brain and the breast is done in control and inhibitor compound treated triple negative breast cancer mice. Western analysis to determine the expression and phosphorylation status of Jak3 and its downstream signaling partner STAT3, and STAT5 is done in brain tissue lysates.

[0101] Anti-TNBC-BM activity of Jak3-targeted compounds

[0102] C1 was administered to NSG mice through IP once every alternate day for 60 days @1 mg / Kg post MDA- 231 -BrM implant into the mammary fat pad. FIG. 8A shows the time course of tumor growth in vehicle control verses C1 and C4. Though, vehicle control group shows growth in tumor volume with time which started to grow significantly after day 15 and continued till 60 days of measurement, treatment with C1 or C4 caused a complete remission in tumor growth at the primary site. Since TNBC tumor growth started after 15 day, in a second set of experiments, primary tumors were resected from the breast tissue, and the mice were treated with C1 for the next 45 days through IP once every day for 45 days @1 mg / Kg post resection. Analysis of TNBC metastasis marker Vimentin in the brain sections of these mice in FIG. 8B shows significant decrease in FITC-labeled anti-vimentin antibodies in C1 treated mice indicative of decrease in brain tumor load in these mice. Quantification of the fluorescent intensities in FIG. 8C and the confocal images in FIG. 8D show that treatment with C1 led to a 60% decrease in brain tumor load compared to the vehicle treated mice. Together, these results show that C1 treatment leads to maintenance of complete remission of tumor at the primary site of breast tissue in 60 days of treatment regimne days and a 60% reduction in metastatic tumor load in the brain during a 45 days treatment regimen.

[0103] To determine the toxicity of the two selected compounds, mice were administrated by intravenous administration an increasing dose of the compounds or vehicle control. During the experiment, their behavior was observed, and the body weight was recorded every day. Loss of body weight, signs of fatigue, and loss of appetite (food intake) was observed. In addition, serum inflammatory markers as a sign of toxicity were also determined. Overall, the data suggested that the mice were able to tolerate up to 10 mg / Kg BW of the compounds. No obvious toxic reaction and loss of body weight was observed after treatment with C1 or C4.

[0104] EXAMPLE 4

[0105] Drug profile analysis of C1 (N-methylsulfonylmethanesulfonamide)

[0106] Jak3-specific dose response

[0107] C1 demonstrates a Jak3-specific dose response in a cell culture model of triple negative breast cancer brain metastases (TNBC-BM). TNBC-BM cells were treated with increasing concentrations of C1 and total tyrosine phosphorylation of Jak1 , Jak2, and Jak3 were determined by immunoprecipitation with pY20 antibodies followed by immunoblotting by respective Jak1 , Jak2, and Jak3 antibodies. Corresponding total Jak1 , Jak2, and Jak3 proteins were determined from the same samples but before immunoprecipitation using respective Jak antibodies. Densitometric analysis of the band intensities were determined by BIORAD GelDoc system, and the ratios of pJak / total Jak are shown. Representative (n=3) blots are shown from 5 independent experiments.

[0108] TNBC-BM cells were treated with increasing concentrations of C1 and total and phosphorylated forms of Jak1 , Jak2, and Jak3 proteins were determined through western analysis. FIGS. 9A-9D show that C1 inhibited tyrosine phosphorylation of Jak3 in a dose dependent manner where at 500nM of C1 Jak3 phosphorylation was almost completely abolished. However, C1 did not affect corresponding tyrosine phosphorylation of either Jak2 or Jak1 at these concentrations. Together, these indicate that C2 had Jak3-specificity for the inhibition of enzymatic activity Jak3 particularly towards its auto-phosphorylation on tyrosine residues. In vivo C1 does not inhibit other Jak kinases in a Jak3-deficient mouse

[0109] Jak3-deficient mice with C1 at the concentration (1 mg / Kg) where C1 showed efficacy towards BM of triple negative breast cancer in an orthotopic xenograft model in NSG mice (see FIGS. 12A-12B) to determine if C1 could inhibit Jak1 or Jak2 in absences of Jak3. FIGS. 10A-1 OB show that in absence of Jak3, C1 did not affect the tyrosine phosphorylation of either Jak1 or Jak2 at the efficacy concentration (FIGS. 12A-12B) of C1 indicating the therapeutic efficacy of C1 was due to specific inhibition of Jak3 and therefore, C1 had Jak3 selectivity over Jak1 and Jak2.

[0110] Similar experiments were done as in FIGS. 10A-10B except using an in vivo system of female Jak3-KO mice treated with 1 mg / Kg of C1 (same efficacy dose as FIGS. 9A-9D) for 6 hours followed of harvesting the hepatic tissues to determine the tyrosine phosphorylation of Jak1 and Jak2 in absence of Jak3. Representative (n=3) blots are shown from 5 independent experiments.

[0111] In vitro C1 demonstrates blood brain barrier (BBB) permeability

[0112] Using transwell endothelial cell-culture system, we determined the BBB permeability of C1. Transwell plates were co-cultured with human endothelial and astrocytes in the epical chamber till confluence followed by treating with C1 (20 mg / ml, final concentration). The BBB permeability was determined through measuring the concentration of C1 in the basolateral bottom chamber at the indicated timepoint using standardized LCMS. Average values with SEM of C1 in the bottom chamber are shown (n=5).

[0113] Determination of C1 concentration in the basolateral chamber in FIG. 11 indicated that C1 was able to cross the in-vitro BBB in a time dependent manner with the highest concentration reached at around 6 h indicating that C1 is BBB permeable. To rule out that these were due to disruption in endothelial junctional proteins, trans-endothelial electrical resistance (TEER) across the membrane of the trans-well remains constant through the duration of the experiments.

[0114] In vivo stimulation of tumoriqenic immune response and in vitro inhibition of proliferation in a dose dependent manner

[0115] C1 stimulates tumorigenic immune response in vivo (FIGS. 12A-12B). Cytokine levels in the serum from C1 treated mice were measured using a mouse Multianalyte cytokine assay kit (Qiagen), and mean values from each group (n=6 per group) are shown, (p 0.05 from at least n=3 independent experiments).

[0116] In vitro proliferation assays demonstrated that C1 effectively inhibits the growth of MDA-MB-231 BrM cells in a dose-dependent fashion. Increasing concentrations of C1 led to a progressive reduction in cell viability, indicating that its antiproliferative effects are directly correlated with drug dosage. These findings suggest that C1 effectively targets key pathways driving the proliferation of metastatic breast cancer cells.

[0117] EXAMPLE 5

[0118] In vivo efficacy of C1 via oral administration in mouse models

[0119] C1 efficacy and pharmacodynamics against development of brain metastases

[0120] Generally, each study includes a vehicle group, a single-agent group with fifteen mice per group and consists of appropriate controls for C1 inhibitors. Mice are monitored and euthanized if they lose 20% of their initial weight, develop ascites, cachexia, or display extreme weakness or inactivity. Body weights and tumor volume measurements are collected every three days. The study is terminated when the vehicle control group reaches 2000 mm3.

[0121] 5-7-week-old female NSG mice (1X1=18, 6 / group) are implanted with 5*106MD-MBA- 231 BR-GFPLuc cells in PBS (100 pL / animal) injected into the fat mammary pad to establish primary TNBC tumors. Mice are treated with vehicle (NS) or C1. Treatments are performed once every 15 days starting on days 0, 15, 30, 45. Animals will be under daily observation for 8 weeks or until their health condition is affected. Body weights and caliper tumor measurements are performed twice a week up until termination at 8-12 weeks.

[0122] To evaluate brain metastases, brain tissues are collected for histopathology analysis to determine metastasis presence. Transcardial perfusion is performed with 5ml 0.9% NaCI followed by 50ml of 4% PFA to preserve brain tissues. Brains will be postfixed in 4% PFA overnight at 8°C (maximum 24h). Tissues are imaged using MRI and quantify metastatic nodules, to finalize model selection based on metastasis characteristics.

[0123] Tumor imaging analysis will be performed on Days 0, 14, 28, 42, and 56. Mice will be lightly anesthetized with isoflurane and then analyzed using the IVIS Spectrum imager. The MD-MBA-231 BR-GFPLuc model mice receive 150 mg / kg of D-Luciferin (Perkin Elmer # 122799) in PBS via an intraperitoneal injection 5-15 minutes prior to image acquisition with an IVIS imager. The ventral images are taken and averaged. Exposure time is adapted to the amount of luciferase activity and D-luciferin in order to optimize signal levels and prevent saturation of the light sensors. Images are analyzed using defined regions of interest (ROIs) using the Life Image from Perkin Elmer software. Bioluminescence data will be expressed as photon flux (Photon s-1 sr-1 ) in the ROL Background radiance is defined from ROI drawn over background. Toxicity is assessed after 28 days by measuring body weight, blood chemistry, complete blood count, and histopathology (hepatic and cardio pathology). Terminal collections are performed for the primary tumor, brains and lungs.

[0124] To evaluate C1 in prevention or inhibition of brain metastases, 1 TNBC PDX model is selected for efficacy studies. Using 24 female NSG mice (N=8 / group) and pre-treating with 1 mg / kg of C1 via oral gavage on D-2., selected PDX tumor slurry (1 OOpL / animal) is implanted in the mammary fat pad on DO. C1 treatment is continued once every 15 days, monitoring animals daily for 8 weeks or until health is affected. Body weights and tumor growth are measured twice weekly up to termination.

[0125] C1 efficacy and pharmacodynamics against a TNBC-BM PDX tumor model

[0126] RNA Seq data for five NexusPharma TNBC PDX models is used to verify gene expression profile of biomarkers. These biomarkers include serine-threonine and tyrosine kinase family members as well as other genes. Based on the RNA expression analysis, from 1 to 6 TNBC PDX models are selected for further evaluation. 5-7-week-old female NSG mice (12 / group) are implanted with PDX tumor slurry (100 pL / animal) injected into the fat mammary pad to establish TNBC tumors. Animals are under daily observation for 8 weeks or until their health conditions is affected. Body weights and caliper tumor measurements are performed twice a week up until termination at 8-12 weeks. Following euthanization, brain tissues are collected and subjected to histopathology evaluation to determine the presence of BM. In addition, at terminal collection, brain tissues are perfused transcardially with 5ml 0.9% NaCI to get the blood out, followed by ice cold 50ml of 4% paraformaldehyde (PFA). Then the brains are postfixed in 4% PFA over night at 8°C (maximum 24h). The tissues are imaged using MRI and the number of metastatic nodules is determined.

[0127] Based on the results from the above step, 1 PDX model is selected for evaluation of efficacy of C1 in oral form for prevention development of brain metastases. To evaluate the efficacy, 24 female NSG mice (N=8 / group) are used, animals are pre-treated on D-2 with 1 mg / kg of C1 using oral gavage, and on DO implanted with the selected PDX tumor tissue slurry in the mammary fat pad (1 OOpl / animal). C1 treatments are continue once every 15 days and animals are under daily observation for 8 weeks or until their health condition is affected. Body weights and caliper tumor measurements are performed twice a week up until termination.

[0128] At terminal collection, brain tissues are perfused transcardially with 5ml 0.9% NaCI to get the blood out, followed by ice cold 50ml of 4% PFA. Then the brains are postfixed in 4% PFA over night at 8°C (maximum 24h). The tissues are imaged using MRI and the number of metastatic nodules will be determined. In addition, primary tumors and lungs are collected and preserved in 10% NBF for further histopathological analysis.

[0129] Results from RNA-Seq, tumor growth analysis, metastasis assessment, and efficacy study are compiled. An unpaired two-sided Student's t-test is used to determine statistical significance between the control and treatment groups. All data is screened for parametric statistical test assumptions. All statistical tests' a priori alpha level is set at p < 0.05.

[0130] EXAMPLE 6

[0131] Effects of C1 in vivo on absorption, distribution, metabolism, excretion (ADME), and blood brain permeability (BBB) Excretion: ADME

[0132] The critical ADME profile of C1 is determined by treating the xenograft mice using TNBC-BM NSG mouse model injected with the cell line MAD 231 -BrM. Both rodent (mouse) and non-rodent (beagle dog) models are used. Based on the toxicity and efficacy studies, a dose range of 1 mg / kg to 100mg / kg was chosen. A single-dose oral or intravenous administration of the C1 drug compound isused. Plasma and tissue samples are collected at multiple time points (0.5, 1 , 6, 12, 24 hours) post-dosing to cover the full PK profile. Drug concentrations in various tissues are measured with a focus on plasma and brain. In addition, liver, heart, kidney, and lungs tissues are taken for analysis. To calculate brain-to-plasma ratio, the concentration of the C1 in the brain relative to plasma is quantified at various time points aiming for a ratio between 0.7 and 1 .0 ratio. This is critical for compounds targeting the TNBC-BM cells metastasized to the brain. LC-MS / MS (liquid chromatography-mass spectrometry) is used with a standardized mass spectrometric detection for high sensitivity and specificity.

[0133] Metabolic stability and elimination

[0134] Studies are conducted in NSG-mice and rats for metabolic stability and elimination. A single dose of the compound is administered both via oral and intravenous routes, as would be administered for human use during FIH trials upon FDA approval. Blood, urine, and feces samples are collected to determine the rate of metabolite formation. C1 concentrations are analyzed over time to confirm that the active ingredient is metabolically stable. In vitro liver microsome studies (human and rat) are performed to investigate the potential for metabolic stability. Liver S9 fractions or recombinant enzymes are used to evaluate the compound's metabolism. Mass spectrometry and NMR (nuclear magnetic resonance) are used to characterize metabolites. Major metabolites are identified to understand their pharmacological activity and stability. For elimination studies, the elimination rate constant (Ke) from plasma samples is measured to determine the compound’s half-life (t! ) and ensure it is consistent with a drug that is completely eliminated within six half-lives. The amount of the parent drug and metabolites in feces and urine is analyzed to quantify the excretion of the compound in feces, urine, and bile for most of the C1 to be excreted in feces (>70% of the dose). Off-target toxicity

[0135] Hepatic, cardiac, and neurological toxicities are determined in mice and beagle dogs are used. C1 is administered via oral gavage using both a single dose and multiple dose regimen to assess the hepatic, cardiac, and neurological toxicities. The biomarkers of liver damage, including ALT (alanine transaminase), AST (aspartate transaminase), bilirubin, and albumin are monitored. Liver histology is performed after necropsy to identify any liver damage or signs of toxicity such as hepatocellular injury or necrosis. Cytochrome P450 (CYP) inhibition studies are conducted to evaluate the potential for the compound to inhibit key cytochrome P450 enzymes, such as CYP3A4, CYP2D6, that may lead to drug-drug interactions. Continuous electrocardiogram (ECG) monitoring is performed for potential arrhythmias. Postmortem cardiac tissue is analyzed for any structural damage to the heart, and circulating biomarkers of cardiac injury, including troponin and B-type natriuretic peptide (BNP), are measured.

[0136] Neurotoxicity assessment

[0137] In vitro studies are used to identify potential neurotoxic mechanisms at the cellular and molecular levels. Primary cultures of human-induced pluripotent stem cell (hiPSC)-derived neurons, glial cells and rodent cortical neurons are used to perform cell viability and apoptosis assays using MTT / XTT assays, Annexin V / PI staining, and caspase activation. In addition, oxidative stress is assessed through ROS generation, mitochondrial membrane potential, and glutathione depletion. For in vivo toxicity assessment, xenograft NSG-mouse models are evaluated for systemic toxicity, neurobehavioral changes, and histopathological effects. Following NSG-mouse model of TNBC-BM, as shown herein, drug administration is performed via IV and neurological functions are assessed. Behavioral and neurofunctional assessments for motor function tests use Rotarod, open-field test, and grip strength. For cognitive function tests, Morris water maze and novel object recognition tests are performed. For Sensory responses, hot plate test and von Frey test are performed for neuropathic pain. For histopathological and neuroinflammatory analysis, H&E and Nissl staining are done to assess neuronal degeneration. Immunohistochemistry for GFAP / lba1 are done to assess astrocyte and microglial activation. ELISA / qPCR for inflammatory cytokines (IL-6, TNF-a, IL-1 ) are performed in brain tissue to assess neuroinflammation.

[0138] BBB permeability

[0139] An organ on chip model of Synvivo’s SynBBB 3D Blood Brain Barrier system is used using the protocol as described by the supplier (Synvivobio). Briefly, SynVivo SynBBB recreates the in vivo microenvironment by emulating brain tissue cells in communication with endothelial cells across the BBB. The microfluidic model has two chambers: a tissue chamber to emulate the brain, and a separate chamber for the vasculature. Human brain microvascular endothelial cells (BMECs) are used. Co-culture of these cell types establishes the BBB in the microfluidic devices.

[0140] Once the model is established, C1 is administered and a drug permeability assay performed. Briefly, FITC-Dextran (positive control) or C1 (fluorescently labeled C1 ) is prepared in serum-free media and perfused into a SynVivo microfluidics device previously coated with fibronectin and seeded with endothelial cells and astrocytes at 0.1 pL / min. A phase image of the device is captured before beginning the assay and fluorescence of the apical and basolateral channels is captured every 30 seconds over 1 hour. Molecule permeability is assessed by video analysis using Imaged and calculated according to Eq. 1 : where Her is the hematocrit count (equal to 0 for in vitro measurements), lvo is the fluorescent intensity in the apical channel containing the endothelial cells, V / S is the ratio of apical volume to surface area (in this case, calculated for a 200 pm wide and 100 pm height vascular channel), and ltis the fluorescent intensity in the basolateral chamber.

[0141] Data analysis

[0142] For the PK study, plasma concentration versus time curves (Cmax, Tmax, AUC) is plotted, and calculate the half-life (t! ), clearance, and volume of distribution (Vd) are calculated. PK software (e.g., WinNonlin, Phoenix) models the drug’s concentration-time curves, determines key PK parameters (e.g., Cmax, Tmax, AUC), and performs noncompartmental or compartmental analysis. A dose-proportionality study is performed to ensure that the PK profile of the compound remains consistent across various doses. For biodistribution, the tissue-to-plasma ratio is computed to ensure that 80% of the drug is distributed into plasma. For elimination studies, the elimination rate constant (Ke) from plasma samples is measured. The compound’s half-life (t! ) is determined to ensure it is consistent with a drug that is completely eliminated within six half-lives.

Claims

WHAT IS CLAIMED IS:

1. An allosteric compound that selectively targets a non-kinase domain in Janus Kinase 3 enzyme (Jak3).

2. The allosteric compound of claim 1 , wherein the non-kinase domain is a Jak3- Src homology 2 (Jak3-SH2) domain.

3. The allosteric compound of claim 1 , comprising N- methylsulfonylmethanesulfonamide, N-(1 -cyano-1 -methyl-ethyl)-2-methoxy- ethanesulfonamide, N-(2-cyanophenyl)methyl-N-ethyl-ethanesulfonamide, N-(2-cyanoethyl)- N-methyl-propane-1 -sulfonamide, or 5-methyl-2-prosylsulfonyl-benzamide or a combination thereof.

4. The allosteric compound of claim 3 that is N- methylsulfonylmethanesulfonamide.

5. A pharmaceutical composition comprising the allosteric compound of claim 1 and a pharmaceutically acceptable diluent or excipient.

6. A method for treating a breast cancer in a patient in need thereof, comprising: administering at least once to the patient an amount of the pharmaceutical composition of claim 5 pharmacologically effective to inhibit at least one activity of a Jak3 enzyme.

7. The method of claim 6, said administering step comprising selectively targeting a Jak3-SH2 domain within the Jak3 enzyme.

8. The method of claim 6, wherein the pharmaceutical composition comprises N- methylsulfonylmethanesulfonamide and the diluent or the excipient.

9. The method of claim 6, wherein the activity of the Jak3 enzyme is a signaling pathway within cells associated with the breast cancer.

10. The method of claim 6, wherein the breast cancer is a triple negative breast cancer (TNBC) or a metastatic cancer thereof.

11. The method of claim 10, wherein the metastatic cancer is a metastatic brain cancer.

12. A method of preventing or reducing the likelihood of a triple negative brain cancer metastasizing in a subject, comprising: administering at least once to the subject a pharmaceutically effective amount of N- methylsulfonylmethanesulfonamide or a pharmaceutical composition thereof.

13. The method of claim 12, wherein the triple negative breast cancer metastasizes to a metastatic brain cancer.

14. A method for inhibiting proliferation of cancer cells overexpressing a Janus kinase 3 enzyme, comprising: contacting the cancer cells with at least one inhibitor selective for a Jak3-SH2 domain in the Janus kinase 3 enzyme.

15. The method of claim 14, wherein the contacting step comprises inhibiting at least one signaling pathway in the cancer cells.

16. The method of claim 14, wherein the inhibitor is at least one of N- methylsulfonylmethanesulfonamide, N-(1 -cyano-1 -methyl-ethyl)-2-methoxy- ethanesulfonamide, N-(2-cyanophenyl)methyl-N-ethyl-ethanesulfonamide, N-(2-cyanoethyl)- N-methyl-propane-1 -sulfonamide, or 5-methyl-2-prosylsulfonyl-benzamide or a combination thereof.

17. The method of claim 16, wherein the inhibitor is N- methylsulfonylmethanesulfonamide.

18. The method of claim 14, wherein the cancer cells comprise a breast cancer, a uterine sarcoma, a uterine endometrial cancer, or an ovarian cancer.

19. The method of claim 18, wherein the breast cancer is a triple negative breast cancer.

20. The method of claim 19, wherein the triple negative breast cancer cells are metastatic brain cancer cells.21 . An inhibitor compound selective for a specific domain in a Janus Kinase 3 enzyme (Jak3).

22. The inhibitor compound of claim 21 , wherein the specific domain is a kinase domain, said inhibitor comprising 3-(1-(5-(4-fluorophenyl)pyrazolo 1 ,5-a)pyrimidin-7- yl)piperdin-3-yl) propanamide, 5-(2-(5-fluoro-1 H-1 ,3-benzodiazol-2-yl)pyrrolidine-1-carbonyl)- n-phenylpyrimidine-2-amine, N-cyclopropyl-N-((1 H-indol-5-yl)methyl-6-(4H-1 ,2,4-triazol-4- yl)pyridine-3-carboxamide, N-(5-)carbam(1 ,5-dimethyl-1 H-pyrazol-4yl)carbamoyl)-ethyl-1 H- pyrazol-4-yl)-1 -methanesulfonyl piperdine-3-carboxamide, N-(1 -2,6-dichlorophenyl)methyl)- 1 H-pyrazol-3-yl)-5-(l-methyl-1 H-pyrazol-4-yl)-1 ,3,4-thiadiazol-2-amine, 4-(azocane-1 - carbonyl -7-fluoro-2-(2-methylpyrimidin-5-5-yl)quinoxaline-4-amine, 1 -(3-chloro-4- fluorphenyl)-4-(4-(2,5-dimethylphenyl)-1 ,3-th iazol-2-yl)-1 H-1 ,2,3-triazol-5-amine, or N-(3- ethoxypropyl)-4(2-(4-methoxyphenoxyl)pyrimidin-5-yl) benzamide, or a combination thereof.

23. The inhibitor compound of claim 21 , wherein the specific domain is a Jak3-SH2 domain, said inhibitor comprising N-(1-cyano-1-methyl-ethyl)-2-methoxy-ethanesulfonamide, N-(2-cyanophenyl)methyl-N-ethyl-ethanesulfonamide, N-(2-cyanoethyl)-N-methyl-propane-1- sulfonamide, or 5-methyl-2-prosylsulfonyl-benzamide or a combination thereof.

24. The inhibitor compound of claim 21 , wherein the specific domain is a FERM- SH2 domain, said inhibitor comprising (25,35)-3-amino-2-hydroxy-pentanedioic acid, 5[(2R)- 3-hydroxy-2-(methylamino)-3-oxo-propyl]-1-methyl-pyrazole-4-carboxylic acid, (3R)-3-amino- 3-(1 H-imidazo [4,5-b]pyridin-2-yl)propanoic acid, (2R,3R)-3-amino-2-hydroxy-pentanedioic acid, or 5[(2R)-2-amino-3-hydroxy-3-oxo-propyl]-1-pyrazole-4-carboxylic acid or a combination thereof.

25. A pharmaceutical composition comprising the inhibitor compound of claim 21 and a pharmaceutically acceptable diluent or excipient.

26. A method for treating a breast cancer in a patient in need thereof, comprising: administering at least once to the patient an amount of the inhibitor of claim 21 or a pharmaceutical composition thereof either of which is pharmacologically effective to inhibit at least one activity of the Jak3 enzyme.

27. The method of claim 26, wherein the breast cancer is a triple negative metastatic breast cancer or a brain metastasis thereof or a combination thereof.

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